A new approach combining angle selected electron nuclear double resonance (ENDOR) spectroscopy with molecular biological techniques to selectively enrich proteins biosynthetically with isotopes will be applied to determine the catalytically competent active site structure of Class A TEM-1 beta- lactamase in substrate hydrolysis. Nitroxy1 spin-labeled beta-lactam derivatives of penicillin and cephalosporin, shown to exhibit high catalytic specificity and reactivity, will be employed as spectroscopic substrate probes of active site structure. The enzyme will be isolated from E. coli growth on perdeuterated algal hydrolyzate as the culture medium. Auxotrophic strains also suitably engineered to overproduce TEM-1 beta-lactamase will be used for growth on deuterated medium to site specifically incorporate isotopically enriched amino acids. A series of four different mutants (E104C, E171C, E240C, and M272C) will be used into which cysteine residues will be engineered within a 6 - 12 A radius of the nitroxyl group as strategic spectroscopic marker probes of active site structure. A series of double mutants will be further developed, each containing one of the mutant cysteine residues and an E166N, R164S, or D179N mutation. The latter two mutations disrupt the hydrogen bonding stabilizing the omega-loop near the active site while the E166N renders the enzyme deacylation defective. Both the Michaelis complex and the acylenzyme reaction intermediate for each mutant species will be cryokinetically stabilized for ENDOR spectroscopy. In addition to characterizing the structural perturbations due to R164S and D179N mutations, the studies will be directed towards determining the orientation of the two types of substrates with respect to other critical residues in the active site in both Michaelis complex and acylenzyme reaction intermediates to assign the protein residue and location of structured water molecules responsible for protonation of the beta-lactam group. The ENDOR distance measurements will be then applied as constraints in molecular dynamics simulations of the corresponding reaction intermediates to assess how active site residues must rearrange in conformation and orientation from that in the free enzyme to assume a catalytically competent structure. Since the R164S mutation confers antibiotic (cephalosporinase) resistance in clinical isolates, the results will also identify critical substrate-protein interactions that are important to understand for improved design of beta-lactamase inhibitors for therapeutic purposes.